D-(−)-3-hydroxybutyric acid
is a very valuable intermediate in fine chemical industry, which can be directly used as drug for treating various diseases (Russia Patent 2096035-C1, U.S. Pat. No. 5,112,865, WO00/28985). The traditional methods for the production of D-(−)-3-hydroxybutyric acid (3HB) are chemical synthesis, and a process involving obtaining the 3HB degrading from its polymer poly-D-(−)-3-hydroxybutyric acid (PHB) which is synthesized by bacteria. Both of these two methods relate to very complicated techniques, require a complex chiral separation process to get the target compound, and require large production input, leading to much higher costs of D-(−)-3-hydroxybutyric acid production. On the other hand, environmental pollution also exists in the process of chemical synthesis and chiral separation. Therefore, there still exists a demand for new methods for the production of D-(−)-3-hydroxybutyric acid, which methods are cost-effective and avoid the chiral separation process.
It has been known that β-ketothiolase encoded by gene phbA (or pbaA) catalyzes two acetyl-CoAs into acetoacetyl-CoA; and that acetoacetyl-CoA reductase encoded by gene phbB (or phaB) catalyzes acetoacetyl-CoA into D-(−)-3-hydroxybutyryl-CoA. The combined use of gene phbA and gene phbB in a bacteria can lead to the production of hydroxybutyryl-CoA. Gene ptb and gene buk encode phosphotransbutyrylase and butyrate kinase, respectively. Enzymes encoded by these two genes can catalyze hydroxybutyryl-CoA into D-(−)-3-hydroxybutyric acid in bacteria. Thus, the inventors attempted to use the combination of these four genes to produce D-(−)-3-hydroxybutyric add via a one-step method in engineered bacteria, and succeeded finally.